1. Field of the Invention
The present invention relates generally to a vaccine, and more particularly, to a sub-unit vaccine comprising structural proteins VP2 and VP3 of Infectious Pancreatic Necrosis Virus (IPNV) assembled as an empty viral capsid.
2. Description of the Related Art
Epizootics of viral infections are devastating in hatcheries and ponds rearing either cold or warm water fish and repeated disease outbreaks can jeopardize the financial survival of an operation. Thus, the health of fish is critical to the survival of the aquaculture industry and effective vaccines are desperately needed.
Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly contagious and destructive disease of juvenile Rainbow and Brook trout and Atlantic salmon. Young fish (two-to four-months old) appear to be the most susceptible to IPNV infection, resulting in high mortality. In trout and salmon, IPNV usually attacks young fry about five to six weeks after their first feeding. The affected fish are darker than usual, have slightly bulging eyes and often have swollen bellies. At the beginning of an outbreak, large numbers of slow, dark fry are seen up against water outflows, and fish are seen “shivering” near the surface. The shivering results from a characteristic symptom of the disease, a violent whirling form of swimming in which the fish rotate about their long axis. If the affected fish are examined, a characteristic white mucus is seen in the stomach. The pancreas appears to be the primary target organ for the virus.
After an IPNV outbreak, the surviving fish generally become carriers of the virus. Trout that are carriers of the virus are a serious problem for the aqua-culture industry because the only control method currently available on a commercial basis for eliminating the virus in carrier fish is destruction of these fish.
Highly virulent strains of IPNV may cause greater than 90% mortality in hatchery stocks in less than four months old. Survivors of infection can remain lifelong asymptomatic carriers and serve as reservoirs of infection, shedding virus in their feces and reproductive products. The virus is capable of infecting a number of different hosts and has a worldwide presence. IPNV can have serious economic consequences for commercial trout and salmon farms and are therefore a major concern within the aquaculture industry. Therefore, IPNV is a pathogen of major economic importance to the aquaculture industry.
IPNV is the prototype of the Birnaviridae virus family. IPNV contains a bisegmented dsRNA genome, which is surrounded by a single-shelled icosahedral capsid. The larger of the two genome segments, segment A (3097 bases), encodes a 106-kDa precursor polyprotein which is processed to yield mature viral structural proteins VP2 and VP3, and VP4 (also named NS) a non-structural protein (Duncan et al. 1987). VP2 has been identified as the major host protective antigen of IPNV. The genome segment B encodes a minor internal polypeptide VP1 (94 kDa) which is the putative virion-associated RNA-dependent RNA polymerase.
An ideal vaccine for IPNV must induce protection at an early age, prevent carrier formation, and should be effective against a large number of IPNV subtypes. One approach has been the use of killed virus as a vaccine. For example, if formalin-inactivated virus is injected intraperitoneally into four week post-hatch fry, the fish becomes immunized (Dorson, J. Virol 21:242-258, 1977). However, neither immersion of the fish into a liquid suspension of killed virus nor oral administration thereof has been found effective. Thus, the main problem with using killed virus is the lack of a practical method for administration for large numbers of immature fish because injection of the vaccine is impractical.
The use of attenuated viral strains have also been used as vaccines. However, the earlier attenuated strains either failed to infect the fish or failed to induce protection. Strains with low virulence have been tested as vaccines for more virulent strains, but mortality from the vaccinating strain was either too high or protection was only moderate (Hill et al., “Studies of the Immunization of Trout Against IPN,” in Fish Diseases, Third COPRAQ Session (W. Ahne, ed.), N.Y., pp. 29-36, 1980).
Recent reports have shown that expression of virus coat proteins often results in self-assembly of virus-like particles (VLP) that are essentially empty whole virions. Of these VLP-producing systems, vaccines have been proposed for poliovirus (Urakawa et al. 1989), parvovirus (Saliki et al. 1992), bluetongue virus (Belyaev et al. 1993) and infectious bursal disease virus (IBDV)—a member of the Birnaviridae family (Vakharia, et al. 1994; Bentley, et al. 1994).
However, several attempts have been made to recreate the same results for IPNV but to date these attempts have not been shown effective for various reasons. For instance, McKenna, et al. 2001 reported that virus like particles were generated through expression of Segment A by recombinant Semliki Forest Virus (SFV). Notwithstanding this alleged outcome, no conclusive proof was presented that the produced virus-like particles were indeed empty viral capsids. Several blots and electron microscopy slides show some type of virus like particles but without substantial proof of the formation of empty IPNV capsids resembling the size and 3D-structure of the native IPNV virus structure.
Magyar and Dobos, 1994 reported cloning of IPNV segment A into baculovirus expression vectors and expressing proteins pVP2, VP4 and VP3 in insect cells. However, as reported by Magyar and Dobos, using the baculovirus expression vectors in the insect cells did not show virus like particles that were correctly processed into a tertiary structure representing an empty viral capsid. Review of the process described in Magyar and Dobos it is clear that generating an empty IPNV capsid was impossible because Magyar and Dobos included the very first ORF of Segment A which encodes the minor 17-kDa nonstructural protein referred to as VP5 which partly overlaps the major ORF of VP2-VP-4-VP3 proteins. The VP5 protein is toxic to the cells and hence affects the production of any of the proteins. Thus, even though the proteins may have been expressed in the insect cells the proteins were not post-translationally modified and correctly folded into an empty IPNV capsid.
Phenix, et al. (2000) describes production of virus-like particles that were generated by expressing the IPNV VP2 protein by means of a Semliki Forest Virus expression vector. However, only the VP2 protein was expressed without expressing the VP3 protein and as such, the correct formation of an empty capsid is not formed. Further, without expression of the protein VP3, aggregates may form but without the correct conformation to form neutralizing epitope. The VP2 aggregates that were formed are smaller (25 nm) than virus-like particles that include a fully conformational folded viral capsid (approximately 50 to 65 nm and typically about 60 nm).
Inactivated IPNV vaccines have been found to be efficacious by intraperitoneal inoculation IPNV (Leong and Fryer 1993). In addition, it was shown that the complete polyprotein of segment A expressed in E. coli induced protective immunity after intraperitoneal inoculation in rainbow trout fry. However, intraperitoneal inoculation for a vaccine delivery method is not very practical and bacteria are not optimal hosts for the production of many types proteins.
Therefore, interest has centered in other eukaryotic protein expression systems, notably yeast and insect cells in culture, as possible hosts for the production of recombinant proteins. For this reason, and related reasons, there has been effort directed toward the tissue culturing of insect cells to produce recombinant proteins. Several systems have been developed for the culture of insect cells in vitro, and vectors have been developed which are capable of transgene expression in insect cells. The transforming vectors are most commonly made from a group of insect pathogenic viruses belonging to the Baculoviridae family, the viruses being known as Baculoviruses. Baculoviruses are characterized by a circular double-stranded DNA genome and a rod-shaped enveloped virion. The DNA can be manipulated to incorporate a gene which encodes a subject protein and the DNA of the baculovirus will cause the cells of its host to produce the proteins encoded in its DNA.
Another approach to the production of recombinant proteins is based on the use of live insect larvae. Such an approach uses, in effect, the insect larvae as a factory for the manufacture of the desired gene product. The transgene can be expressed in the larvae through the baculovirus expression system, allowed to proliferate, and then recovered from the larvae. Because insect larvae can be grown quickly and inexpensively and the yields obtained from insect larvae is greatly increased relative to that obtained from bacterial cells makes them an appealing alternative to cell based protein manufacturing.
Attie et al., U.S. Pat. No. 5,472,858 disclosed this approach with the tobacco hornworm. After the hornworm is infected with a recombinant baculovirus, it begins secreting the recombinant protein into its hemolymph. The hemolymph can then be withdrawn using a syringe throughout the larvae's growth. However, there is a drawback to this specific method. Although the tobacco hornworm larvae is ideal for the physical manipulation because of its large size, a great deal of manual labor is required to extract the recombinant protein if large numbers are to be cultivated.
Accordingly there is a need for an IPNV sub-unit vaccine and method of producing same that overcomes the shortcomings of the prior art, that does not exhibit the problems related to live vaccine and/or attenuated vaccines, can be easily produced and recovered, and the proteins that are expressed are post-translationally modified and correctly folded into the conformation structure that exposes neutralizing epitopes.